Chromatography Forum

I am going to analyze blood serum for 25-OH D2 and 25-OH D3.
I found this application note which i am going to follow through.
There is one thing that i can not quite understand. And that is why they are using a Cation Exchange SPE column.. As far as i can see 25-OH D2 and 25-OH D3, are containing Oxygen groups and are hard to ionize to 1+. So how can the vitamin be purified with a cation?
My first thought is to raise the pH and use a Anion exchanger, and lower the pH to eluete my vitamin.

Structure of vitamin D2 and D3:


What do you guys think?

- Adrian

This mechanism is not Cation exchange, just reverse phase mechanism.

6. Sequentially wash the column with 1x1 mL methanol : water (50:50), then 1x1 mL methanol : water (75:25), using gravity flow.
7. Turn the vacuum on for 30 sec, and then add 200 μL of n-heptanes (Scientific Products) to each column. Let the n-heptanes sit for 2 min, then apply full vacuum for 5-7 min.
8. Elute with 250 μL of methanol under gravity flow only for 2 min, followed by 250 μL of deionized water.

INGREDIENTS of Multiprep® Extraction Columns, GV-65C and GV-130C,
Components: Divinylbenzene/Styrene copolymer (Proprietary Formulation) and sodium bisulfite

Thanks! I misunderstood the Multiprep column. I thought it was a cation

Dear Adrian,
Just a quick question.

did you manage to reproduce the method?

Thanks,
Carol